Rodent cells have provided a useful tool for the analysis of cellular factors that are important for HIV-1 entry and transcription. Indeed, the expression of certain human genes in rodent cells is able to rescue rodent-cell specific blocks in these processes. However, despite expression of all species-specific co-factors known to be required for HIV-l replication, most rodent cells remain unable to efficiently support even a single cycle of HIV- 1 replication. In fact, we have observed a profound defect in HIV particle production by many rodent cells and the few particles that are produced are significantly less infectious than those derived from human cells. In the studies detailed in this proposal, we seek to determine how viral and species-specific host-cell factors interact to mediate infectious particle production. The defective phenotype in rodent cells often correlates with a reduced level of intracellular processing of the Pr55Gag precursor, a finding which suggests that Pr5SGag targeting to the correct site of virion assembly is the underlying defect. The biochemical and microscopic imaging experiments outlined herein will directly test this hypothesis and will further examine whether this phenotype is a consequence of a failure of rodent cells to support appropriate post-translational modifications of Pr55Gag. Experiments will also be done that determine whether rodent cell derived virions exhibit any defect in protein or RNA composition. Since the matrix region of Pr55Gag contains the major determinants of subcellular localization, a genetic analysis will focus on this domain. Specifically, we will determine whether defective particle formation in rodent cells is a general property of lentiviruses and examine whether redirecting Pr5SGag to the plasma membrane of rodent cells by manipulation of matrix sequences is sufficient to restore extracellular particle formation and infectious virus production. The finding that infectious virus production by rodent cells can be substantially rescued by fusion with human cells indicates that components of the human host-cell must play a positive and specific role in this process. We will determine whether single or multiple human chromosomes are required to restore infectious virus production in the context of a rodent cell and we will perform a genetic screen designed to identify human co-factors that enhance infectious HIV-l particle production by rodent cells. These studies have the potential to reveal new targets for chemotherapeutic intervention in HIV disease and may make a rodent model of HIV infection feasible.